Valerie Laval

Development of a Real-Time PCR Method for the Differential Detection and Quantification of Four Solanaceae in GMO Analysis: Potato (Solanum Tuberosum), Tomato (Solanum Lycopersicum), Eggplant (Solanum Melongena), and Pepper (Capsicum Annuum)

The labeling of products containing genetically modified organisms (GMO) is linked to their quantification since a threshold for the presence of fortuitous GMOs in food has been established. This threshold is calculated from a combination of two absolute quantification values: one for the specific GMO target and the second for an endogenous reference gene specific to the taxon. Thus, the development of reliable methods to quantify GMOs using endogenous reference genes in complex matrixes such as food and feed is needed. Plant identification can be difficult in the case of closely related taxa, which moreover are subject to introgression events. Based on the homology of beta-fructosidase sequences obtained from public databases, two couples of consensus primers were designed for the detection, quantification, and differentiation of four Solanaceae: potato (Solanum tuberosum), tomato (Solanum lycopersicum), pepper (Capsicum annuum), and eggplant (Solanum melongena). Sequence variability was studied first using lines and cultivars (intraspecies sequence variability), then using taxa involved in gene introgressions, and finally, using taxonomically close taxa (interspecies sequence variability). This study allowed us to design four highly specific TaqMan-MGB probes. A duplex real time PCR assay was developed for simultaneous quantification of tomato and potato. For eggplant and pepper, only simplex real time PCR tests were developed. The results demonstrated the high specificity and sensitivity of the assays. We therefore conclude that beta-fructosidase can be used as an endogenous reference gene for GMO analysis.

Interactions between Head Blight Pathogens: Consequences for Disease Development and Toxin Production in Wheat Spikes

ABSTRACT Head blight (HB) is one of the most damaging diseases on wheat, inducing significant yield losses and toxin accumulation in grains. Fungal pathogens responsible for HB include the genus Microdochium, with two species, and the toxin producer genus Fusarium, with several species. Field studies and surveys show that two or more species can coexist within a same field and coinfect the same plant or the same spike. In the current study, we investigated how the concomitant presence of F. graminearum and another of the HB complex species influences the spike colonization and the toxin production by the fungi. To study these interactions, 17 well-characterized isolates representing five species were inoculated alone or in pairs on wheat spikes in greenhouse and field experiments. The fungal DNA in the grains was estimated by quantitative PCR and toxin contents (deoxynivalenol and nivalenol) by ultraperformance liquid chromatography-UV detection-tandem mass spectrometry. The responses of the different isolates to the presence of a competitor were variable and isolate specific more than species specific. The development of the most aggressive isolates was either unchanged or a slightly increased, while the development of the less aggressive isolates was reduced. The main outcome of the study was that no trend of increased toxin production was observed in coinoculations compared to single inoculations. On the contrary, the amount of toxin produced was often lower than expected in coinoculations. We thus conclude against the hypothesis that the co-occurrence of several HB-causing species in the same field might aggravate the risk linked to fusarium toxins in wheat production.

Mutual Exclusion between Fungal Species of the Fusarium Head Blight Complex in a Wheat Spike

ABSTRACT Fusarium head blight (FHB) is one of the most damaging diseases of wheat. FHB is caused by a species complex that includes two genera of Ascomycetes: Microdochium and Fusarium. Fusarium graminearum, Fusarium culmorum, Fusarium poae, and Microdochium nivale are among the most common FHB species in Europe and were chosen for these experiments. Field studies and surveys show that two or more species often coexist within the same field or grain sample. In this study, we investigated the competitiveness of isolates of different species against isolates of F. graminearum at the scale of a single spike. By performing point inoculations of a single floret, we ensured that each species was able to establish independent infections and competed for spike colonization only. The fungal colonization was assessed in each spike by quantitative PCR. After establishing that the spike colonization was mainly downwards, we compared the relative colonization of each species in coinoculations. Classical analysis of variance suggested a competitive interaction but remained partly inconclusive because of a large between-spike variance. Further data exploration revealed a clear exclusion of one of the competing species and the complete absence of coexistence at the spike level.

Optimized real-time qPCR assays for detecting and quantifying the Fusarium and Microdochium species responsible for wheat head blight, as defined by MIQE guidelines

In order to better understand the Fusarium head blight disease, reliable real-time PCR assays for detection and quantification of fungal species belonging to the Fusarium and Microdochium genus are needed. Specific qPCR assays were developed for nine of those species. All criteria required for reproducing the assays are presented. The assays were species specific and allow quantification of at least 5 pg of fungal DNA and detection of 0.5 pg of fungal DNA per PCR reaction. Moreover we showed that the quantification performances of the tests were not altered in the presence of DNA of closely related species in the sample. The assays were tested on field samples and have been already used in greenhouse experiments.Reliable real-time PCR assays for detecting and quantifying fungal species of the genera Fusarium and Microdochium are required, to improve our understanding of Fusarium head blight disease. We developed specific qPCR assays for nine species from these two genera. All the criteria required for assay reproducibility are presented. These assays are species-specific and have a quantification limit of 5 pg of fungal DNA/PCR and a detection limit of 0.5 pg of fungal DNA/PCR. Furthermore, the quantification performances of the tests were not affected by the presence of DNA from closely related species. The assays were tested on field samples and have already been used to analyze greenhouse experiments.

Crop residues in wheat-oilseed rape rotation system: a pivotal, shifting platform for microbial meetings

Crop residues are a crucial ecological niche with a major biological impact on agricultural ecosystems. In this study we used a combined diachronic and synchronic field experiment based on wheat-oilseed rape rotations to test the hypothesis that plant is a structuring factor of microbial communities in crop residues, and that this effect decreases over time with their likely progressive degradation and colonization by other microorganisms. We characterized an entire fungal and bacterial community associated with 150 wheat and oilseed rape residue samples at a plurennial scale by metabarcoding. The impact of plant species on the residue microbiota decreased over time and our data revealed turnover, with the replacement of oligotrophs, often plant-specific genera (such as pathogens) by copiotrophs, belonging to more generalist genera. Within a single cropping season, the plant-specific genera and species were gradually replaced by taxa that are likely to originate from the soil. These changes occurred more rapidly for bacteria than for fungi, known to degrade complex compounds. Overall, our findings suggest that crop residues constitute a key fully-fledged microbial ecosystem. Taking into account this ecosystem, that has been neglected for too long, is essential, not only to improve the quantitative management of residues, the presence of which can be detrimental to crop health, but also to identify groups of beneficial micro-organisms. Our findings are of particular importance, because the wheat-oilseed rape rotation, in which no-till practices are frequent, is particularly widespread in the European arable cropping systems.

Stability in the genetic structure of a Zymoseptoria tritici population from epidemic to interepidemic stages at a small spatial scale

Subpopulations of the wheat pathogen Zymoseptoria tritici (26 sample groups composed of 794 pure strains) were collected in two nearby wheat fields, during both epidemic and interepidemic periods of three successive years (2009–2013). The alternative presence of wheat debris allowed taking into account different forms (ascospores vs. pycnidiospores) of inoculum and, thus, its putative origin (local vs. distant). We used a molecular epidemiology approach, based on population genetic indices derived from SSR marker analysis. The aim was to describe putative changes in the neutral genetic structure and diversity of these subpopulations over the time-course of several epidemics, in two close fields, i.e. at a spatiotemporal scale consistent with epidemiological observations, and to determine the possible structuring factors. Genetic structure was stable over time (within and between years) and between fields. All subpopulations displayed very high levels of gene and genotypic diversity. The low levels of linkage disequilibrium and the low clonal fraction, and the similar frequencies of the two mating types in most subpopulations were consistent with the regular occurrence of sexual reproduction in the two fields over the epidemic and interepidemic stages. In conclusion, at this fine spatiotemporal scale, we found that the period, the nature of inoculum and its putative origin had little effect on the short term evolution of the local population, with population size and diversity apparently large enough to prevent genetic drift, and with a major contribution of migration between and within plots to the stabilization of genetic diversity.